Animal Learning & Behavior /998,26 (4),397-407

Relationship ofnegative contrast to animal models offear and anxiety

CHARLES F,FLAHERTY, ANNAGREENWOOD, JOSEPH MARTIN, and MARY LESZCZUK Rutgers University, Piscataway, New Jersey

The relatedness of behavior elicited by reward reduction (successive negative contrast procedure) and behaviors produced by three animal models of anxiety (open-field emergence, elevated plus-maze, and context-shock fear conditioning) was examined by correlational and factor analytic procedures, Factor analysis (oblique rotation) indicated substantial independence among the tests: Trials 1 and 2 of the plus-maze loaded on two different factors unaccompanied by any other test; open-field emer­ gence and context-shock fear loaded on the same factor; and negative contrast loaded on a fourth fac­ tor. However, negative contrast proved to be a dynamic process, with factor loadings changing across a 4-day postshift period-moving from an independent loading on the 1st postshift day to being clus­ tered with context-shock fear and open-field emergence on the 2nd and 3rd postshift days to being clustered with just context-shock fear on the last postshift day. These latter datasupport a multistage theory ofsuccessive negative contrast.

Rats shifted from a preferred reward to a less preferred We have recently proposed a multistage theory ofSNC reward show an abrupt decrement in goal-directed behav­ that suggests that perceptual and cognitive stages (detec­ ior and an increase in exploratory behavior (e.g., Crespi, tion that the new reward is different from the memory of 1942, 1944; Elliott, 1928; Flaherty, 1996). The goal­ the preshift reward; hedonic evaluation ofthe new reward; directed performance of the shifted rats decreases to a search for the "missing" reward if the new reward is of level below that of unshifted control animals given the lesser value) precede the activation of an emotional re­ same level of reward-an outcome termed a successive sponse to reward reduction (Flaherty, 1996). In the present negative contrast (SNC) effect. paper, we are concerned with an investigation ofthis hy­ One enduring interpretation of successive negative pothesis, and we compare daily postshift performance of contrast is that it is caused by an emotional reaction to the rats downshifted in reward with their performance in three reward reduction. Terms such as anger, depression, dis­ procedures frequently used to assess anxiety or fear in rats: appointment, andfrustration have often been used in re­ the elevated plus-maze, context-shock fear conditioning, ferring to a rat's or a monkey's reaction to reduced reward and emergence from the home cage to an open field. ie.g., Amsel, 1992; Crespi, 1942, 1944; Flaherty, Krauss, Correlational and factor analytic approaches to animal Rowan, & Grigson, 1994; Gray, 1982, 1987; Spence, models ofanxiety have been used in a number of recent 1956), Evidence supporting the involvement of some papers. Belzung and Le Pape (1994) examined the cor­ form ofstress in negative contrast includes the success of relations in behavior among five different novelty related antianxiety agents (e.g., benzodiazepines, ethanol, bar­ tests: light/dark choice, elevated plus-maze, holeboard, biturates, and morphine) in alleviating aspects ofcontrast free exposure to a novel space, and confrontation with a (Becker & Flaherty, 1982; Flaherty, 1991b, 1996), the el­ novel object. The authors concluded the five different tests evation ofcorticosteroids as a consequence ofreward re­ ofnovelty induced anxiety were not measuring the same duction (Flaherty, Becker, & Pohorecky, 1985; Goldman, psychological state. In a factor analytic study ofdefensive Coover, & Levine, 1973; Mitchell & Flaherty, 1998), co­ behaviors in mice, Griebel, Blanchard, and Blanchard variation of contrast and other forms ofemotional reac­ (1996) concluded that there were five different aspects of tivity in rats selectively bred for performance in avoid­ anxiety measured by their 17 variables. Other studies have ance learning (Flaherty & Rowan, 1989), and the role of also raised the issue that different tasks or test procedures amygdala lesions in reducing contrast (Becker, Jarvis, may be measuring different aspects ofanxiety (File, 1991, Wagner, & Flaherty, 1984). 1995; Handley, McBlane, Critchley, & Njung'e, 1993; Rodgers, Cole, Aboualfa, & Stephenson, 1995). In some cases, a single test may measure multiple components of This research was funded by NIH Grant MH48835 and by funds from anxiety. Exposure ofmice to a single 5-min trial in an el­ the Dean of Arts and SCIencesat Rutgers to the first author, The assis­ evated plus-maze may yield from two to six factors, de­ tance of Cynthia Coppotelli and Matthew Kelsey is greatly appreciated, pending on the number and nature ofdependent variables Correspondence should be addressed to C. F.Flaherty, Psychology De­ partment, New Brunswick campus, Rutgers University. 152 Freling­ scored (Rodgers & Johnson, 1995). However, the sub­ huysen Rd., Piscataway, NJ 08854-8020 (e-mail: tlaherty@psycho-b. stantial effectiveness ofthe benzodiazepine tranquilizers rutgers.edu). in many animal models ofanxiety suggests that there must

397 Copyright 1998 Psychonomic Society, Inc, 398 FLAHERTY, GREENWOOD, MARTIN, AND LESZCZUK

also be some degree ofcommonality (Davis, 1991; Fla­ suppress open-arm entries (Handley et al., 1993; Pellow herty, 1991a, 1991b; Howard & Pollard, 1991; Lister, & File, 1986; Rodgers et aI., 1995). 1991). Behavior in a second trial in the elevated plus-maze In comparing negative contrast with other models of differs from that seen on the first trial. Benzodiazepines anxiety, we used a negative contrast procedure that in­ have been found to be ineffective in the second trial, re­ volved the consumption of sugar solutions. Rats were gardless of whether they were administered on the first given access to a 32% sucrose solution for 5 min a day trial, leading to the suggestion that the second trial mea­ for 10 days, and, on the 11th day, shifted to a 4% sucrose sures a different type ofanxiety than the first trial (File, solution. Typically, the "shifted" rats lick substantially 1990). This has been supported by factor analytic stud­ less ofthe 4% solution than do "unshifted" controls, but ies that found that the measures ofTrial 1 load on a dif­ the shifted rats recover from contrast after approximately ferent factor from those ofTrial 2 (e.g., Fernandes & File, 4 days of access to the new solution (Flaherty, Krauss, 1996; File, Zangrossi, Viana, & Graeff, 1993). Rowan, & Grigson, 1994). The involvement ofsome as­ Context-shock fear conditioning differs from the above pect ofanxiety or stress in negative contrast is indicated models in that it involves a physically aversive event. by the fact that corticosterone levels are elevated after Corticosterone levels have been shown to be elevated after the 2nd, but not the 1st, postshift day (Flaherty et aI., 1985; exposure to the context in which shocks had been given Mitchell & Flaherty, 1998) and that chlordiazepoxide (Urban, Van de Kar, Lorens, & Bethea, 1986), and benzo­ and ethanol reduce contrast when administered acutely diazepines disrupt conditioned fear (Fanselow & Helm­ on the 2nd postshift day, although they are not effective stetter, 1988; Sanger & Joly, 1985), whereas anxiogenics on the 1st postshift day (Flaherty, 1996; Flaherty, Grig­ enhance conditioned fear (Fanselow, Helmstetter, & Cal­ son, & Lind, 1990). cagnetti, 1991). The test battery selected for comparison with contrast The principal purpose of this experiment was to de­ in the present experiment included a measure ofnovelty­ termine whether behavior at different stages ofthe con­ induced anxiety (emergence from the home cage to an trast recovery cycle was differentially correlated with open field), conditioned fear (context-shock pairings), these tests. A secondary purpose was to determine the and a test that involved both generalized anxiety and a relatedness ofthe other three tests themselves. specific fear ofopen places (elevated plus-maze). Previ­ ous studies have investigated novelty-induced anxiety by METHOD measuring latency to emerge from a familiar area to an unfamiliar area (Corey, 1978; Hughes, 1968; Maren, Subjects Patel, Thompson, & Mitchell, 1993), such as from the Sixty male Sprague-Dawley rats purchased from Harlan were animal's cage to a table top (Henck, Mattsson, Rezabek, used as subjects. The rats were approximately 90 days old, weigh­ Carlson, & Rech, 1994), runway (King, 1968), or open ing 300-350 g at the beginning ofthe experiment. The animals were field (Jellestad, Markowska, Bakke, & Walther, 1986). individually housed under 14:lO-h light:dark conditions. The rats were given free access to water and were maintained at 82% oftheir The evidence that exposure to novel context induces anx­ free-fed weight by once-daily feedings. The deprivation regimen is iety in laboratory animals includes increased heart rate, standard for negative contrast experiments. increased defecation, and increased corticosterone release The rats were run in two consecutive groups of30 each, approx­ (Blanchard, Kelley, & Blanchard, 1974; Crawley, 1985; imately I h months apart. Thirty ofthe subjects were subsequently Misslin & Cigrang, 1986). In these tests, antianxiety drugs used in an anticipatory contrast study and then underwent fear con­ increase ambulation (Treit, 1985), increase exploration ditioning. The fear conditioning, but not the anticipatory contrast, data are included in this report. (Crawley, 1985), and decrease emergence latencies into One rat died prior to the beginning of testing. a novel environment (Einon & Tye, 1975). An alternative way to measure novelty-related anxiety Apparatus is through the use of an elevated plus-maze (Fernandes Negative contrast procedure. All testing was done in six Plex­ & File, 1996; File, 1996; Hogg, 1996; Lister, 1987; Pel­ iglas chambers measuring 29 X 28.5 X 32 em. Sucrose was pre­ low, Chopin, File, & Briley, 1985). In this apparatus, ro­ sented in Nalgene test tubes with metal ball-bearing spouts. The dents show fewer entries into open arms (no walls) than tubes were advanced by motors into holes centered on one wall, into closed arms (walls)-an outcome that primarily re­ 6 em above a hardware cloth floor. Licks were measured through a contact relay circuit and were recorded by microprocessors. flects rodents' aversion to open spaces (Pellow et al., Elevated plus-maze. The maze consisted oftwo open arms and 1985; Treit, Menard, & Royan, 1993). Rats show typical two enclosed arms, each measuring 49.5 X 10 em, with black Plex­ stress reactions when placed in an elevated plus-maze, in­ iglas floors. The open arms were bounded by I-em-high ledges on cluding increased corticosterone levels, increased freez­ the sides, but there were no ledges at the ends of the arms. The ing, and increased defecations-more so in open arms closed arms had 39.5-cm-high transparent Plexiglas walls. The than in closed arms (File, Zangrossi, Sanders, & Mabbutt, maze was elevated 50.5 em from the ground. The rats were carried 1994; Pellow et al., 1985). Anxiolytic drugs adminis­ to the room in a clear Plexiglas box measuring 20 X 26 X 19.5 em, which was covered in black paper. The room was brightly lit, and tered on the first trial increase entries into, and time in, white noise was on in the background during testing. A camera was open arms (Pellow et al., 1985). Anxiogenic compounds, mounted directly above the maze (WAT-902A, Edmund Scientific) such as benzodiazepine inverse agonists and yohimbine, and was connected to a monitor in an adjacent room. CONTRAST AND ANXIETY 399

Home-cage emergence. Emergence was tested from the sub­ prior to the start offear conditioning. During acquisition training, the ject's home cage into an open field. Each home cage measured 24.5 rats were transported individually to the testing apparatus and given X 20.5 X 18.5 em and had a wire-mesh bottom and front and solid six trials of 2-sec shocks presented 4 min apart. Approximately stainless steel sides and back. Cages were placed into a circular 30 sec after the last shock presentation, the rats were removed and white Plexiglas open field 76 cm in diameter, with walls 31.5 cm returned to their home cages. The apparatus was cleaned thor­ tall. The experimental room was brightly lit, with white noise in the oughly after each subject. For 20 subjects, training occurred on the background. 6th day after deprivation; for the remaining 10 subjects, training oc­ Fear conditioning. The training and testing was conducted in a curred on the 11th day after deprivation. 23 X 22 X 25.5 em operant chamber, with clear Plexiglas walls and Testing for fear ofthe context occurred on the day after training. a floor consisting ofmetal rods. The chamber was outfitted with shock The rats were again brought individually into the testing room and presentation capabilities and was enclosed in a sound-attenuating were placed into the same apparatus as was used for training. They chamber. A house light was on during training and testing. The foot were given a 5-min test period, during which no shocks were pre­ shock was a 0.8-mA scrambled alternating current presented for sented. Freezing episodes (as described in File, 1990) were 2 sec. recorded every 5 sec in a time-sampling procedure, and proportion of episodes during which freezing occurred was calculated. The Procedure number ofdefecations was also noted. The rats were divided into six groups (for five ofthe six, n = 10; for the remaining group, n = 9) for purposes of counterbalancing Data Analysis the order of administration ofthe first three tests (successive nega­ Initial analyses performed included an analysis ofvariance, fol­ tive contrast, elevated plus-maze, open-field emergence). Thus, all lowed by Fisher's LSD post hoc tests, paired comparison t test, and possible sequences of these tests were administered. There were normal curve analysis using the Shapiro-Wilk W test. Subsequent 2 days between each test, during which the subjects were weighed analyses included Pearson's correlation coefficient (r) on z scores and fed. and a factor analysis using the SAS statistical package with a pro­ Fear conditioning, which was administered to a subset of30 sub­ max rotation method. jects, occurred approximately 60 days after the first three tests had been completed. Thus, fear conditioning was not included in the RESULTS counterbalancing, which applied only to the first three tests. Negative contrast procedure. The animals received 10 days of acquisition training, during which all subjects received access to The results will be presented first in terms ofperfor­ 32% sucrose solution for 5 min daily. On the 11th through 14th mance on each ofthe four tests (negative contrast proce­ days, all subjects received 4% sucrose. dure, elevated plus-maze, home-cage emergence, and con­ No unshifted 4% control group was included, since the interest ditioned fear) and then in terms of relationships among was in degree of decrement in consummatory behavior in shifted the tests as determined by correlational analyses and fac­ rats as a variable to correlate with other measures of anxiety. Lick tor analysis. frequency was presented in order to provide a comparison with the many earlier studies that used this variable and also included un­ shifted control groups. Negative Contrast Procedure Negative contrast was reported as shift percentage on the first Descriptive statistics for this, and all other tests, are postshift day (100- [first postshift day lick frequency/terminal presented in Table 1. The degree ofreduction in lick fre­ preshift day lick frequency]). This measure was used as the index quency in individual animals following the reduction in of the initial reaction to reward reduction. For purposes of assess­ sucrose concentration ranged from 44% to 89%. The dis­ ing the possible changing nature ofcontrast across the postshift pe­ tribution did not differ reliably from a normal distribution riod, shift percentage was also calculated for each ofthe 4 postshift days and entered into a factor analysis with measures from the other (Shapiro-Wilk Wtest,p = .064). Although an unshifted tests. The slope ofthe recovery oflick frequency across the 4 post­ shift days was used as an index of rate of recovery from contrast. Table 1 Sucrose solutions were mixed from commercial grade sugar on Descriptive Statistics for Selected Variables a weight basis (sugar/[sugar + water]) approximately 24 h before of the Tests of Anxiety each experimental session. Variable M SD Range Elevated plus-maze. The rats were carried individually to the experimental room, where they were kept in the carrying box for Negative Contrast Test 5 min prior to the start ofthe test. Subsequently, the rats were placed Percent shift (%) 68 12 44-89 in the center ofthe maze facing an open arm to begin a 5-min trial. Video recordings of the session were later scored in terms of time Home-Cage Emergence Test spent in open and closed arms and number ofentries into open and Two-pawlatency (sec) 124.7 145.1 1-960 closed arms. An entry consisted ofall four paws in a particular arm. Four-pawlatency (sec) 319.6 224.6 10-960 Each subject was given two 5-min trials spaced 72 h apart. Plus-Maze Trial 1 Home-cage emergence. Each rat was carried individually in its Proportion of time in open arms .29 .15 0-.65 home cage to the experimental room, where the cage was turned on Proportion open-arm entries .36 .13 0-.67 its side (the rat was held by the experimenter during the turning) and Total entries 14.9 3.9 6-22 then placed in the center of the open field, and a timer was started. Plus-Maze Trial 2 Time for the subjects to emerge with two paws and then with four Proportion of time in open arms .17 .13 0-.52 paws from the home cage was recorded by an experimenter seated Proportion open-arm entries .24 .14 0-.48 outside ofthe testing room. Maximum time allowed was 16 min. Total entries 16.4 4.1 1-26 Fear conditioning. The animals were on a free-feeding sched­ Conditioned Fear Test ule during the 60-day period following the completion ofthe other three tests and were reduced to 82% of their new free-feeding weight Proportion of time freezing .45 .17 .15-.85 400 FLAHERTY, GREENWOOD, MARTIN, AND LESZCZUK

control group was not included in this experiment, the closed arms (M= 215.4 sec) [t(57) = 17.18,p < .01], shift ratio (1 - [shift percent! 100)) obtained (.32) was open entries (M = 4.1) were fewer than closed entries approximately equivalent to that obtained in standard con­ (M = 12.3) [t(57) = 10.22,p < .01], proportion oftime trast experiments (e.g., Flaherty, Krauss, & Hill, 1994), in open arms was less than proportion of time in closed indicating a typical negative contrast effect. arms [t(57) = 10.87,p < .001], and proportion ofentries Although there were no differences in initial reaction into open arms was less than proportion of entries into to reward reduction among the counterbalancing condi­ closed arms [t(57) = 8.13,p < .001]. tions (F < 1.0), recovery from contrast was affected by Although Trials 1 and 2 were spaced 72 h apart, there prior experience in that the two groups exposed to con­ were substantial changes in the direction offurther avoid­ trast first were slower to recover than were groups that ance of the open arms on Trial 2. Thus, time in open were exposed to contrast second or third [group X day, arms and number ofopen arm entries were reliably lower F(10,94) = 3.88,p < .01]. Simple effects tests indicated on Trial 2 than on Trial 1 [F(1,53) = 40.59,p < .01, and that the groups that had other experiences prior to con­ F(l,53) = 14.64,p < .01, respectively]. Higher on Trial 2 trast did not differ from each other. These data are illus­ than on Trial 1 were time in closed arms [F(I,53) = 34.57, trated in Figure 1, with the two groups that received con­ P < .01], entries into closed arms [F(I,53) = 46.54, P < trast first combined and the four groups that experienced .01], and total entries [F(1,53) = 11.88,p < .01]. Lower contrast second or third also combined. Whether this dif­ on Trial 2 than on Trial I were proportion oftime spent ference in rate ofrecovery was due specifically to expe­ in open arms [F(l,53) = 39.54,p < .01] and proportion of rience in the other test procedures or was simply due to the entries made into open arms [F(1,53) = 40.08,p < .01]. increased amount ofhandling that the animals received There were no overalleffects across trials due to prior ex­ cannot be determined from this study. perience with other tests [number ofopen entries, F(5,53) = 1.l0,p> .36; number ofclosed entries, F(5,53) = 1.07, Elevated Plus-Maze p> .39; time spent in open arms, F(5,53) = 1.07,p> .39; In Trial 1,time in open arms (M = 73.2 sec) was reliably time spent in closed arms, F(5,53) = 1.58, P > .18; pro­ less than time in closed arms (M = 181.6 sec) [t(57) = portion oftime spent in open arms, F(5,53) = 1.l8,p > 10.5, P < .01], entries into open arms (M = 5.5) were .33; and proportion ofentries that were open, F < 1.00]. fewer than entries into closed arms (M = 9.4) [t(57) = However, there were some group X trial interactions 8.25, p < .0 I], proportion of time in open arms was less that showed that changes in behavior that occurred from than proportion of time in closed arms [t(57) = 19.17, the first to the second trial in the plus-maze were influ­ P < .001], and proportion ofentries into open arms was enced by prior test experience. The reliable effects were less than proportion ofentries into closed arms [t(57) = on time in open arms [F(5,53) = 3.92,p < .01], time in 14.58, P < .001]. These data are presented in Figure 2. closed arms [F(5,53) = 3.36,p < .01], and open-arm en­ The same measures also differed reliably in Trial 2. tries [F(5,53) = 2.61,p < .03]. In general, these interac­ Time in open arms (M = 43.5 sec) was less than time in tions indicated that Trial 1versus Trial 2 differences were greater in the rats that experienced the plus-maze first in the sequence oftests. Thus, these interactions indicated 1500 a tendency ofprior test experience (or perhaps handling) >. Contrast Second or Third to reduce the psychological difference between the two u c trials in the plus-maze (see Hogg, 1996). G) ~Q 5- 1000 Q Home-Cage Emergence ~ Emergence from the home cage into an open field

~ Q/.>' showed an extensive range of individual differences for o ~! 1~960 :.J both two-paw latency (range = sec) and four-paw 500 latency (range 10-960 sec). Sixteen minutes (960 sec) c ~! Contrast First = nl was the maximum time allowed; 3 rats failed to emerge G) :E with four paws, and 1 ofthese rats also failed to emerge with two paws. The distributions differed from normal and were skewed toward the lower times to emerge (Shapiro­ 10 11 1312 14 Wilk Wtest,p < .01). There were no significant differences in latency to emerge due to previous experience (Fs < 1.00). Days Figure 1. Lick frequency of rats shifted from 32% sucrose Fear Conditioning (Days 1-10) to 4% sucrose (Days 11-14). The function labeled There was a range ofindividual differences in percent contrastfirst represents the data ofthe 20 rats that received con­ trast testing prior to any other test. The other function represents oftime spent freezing in the shock context (15%-85%), the 40 rats that received contrast after one or both of the open­ and the distribution of these data was not significantly field emergence and elevated plus-maze tests. different from a normal distribution (Shapiro-Wilk W test, CONTRAST AND ANXIETY 401

2!50 Open Arms 2!50 Clo.ed Arms 10 Open Arms

200 200 10.1 -c.i -c.i F= 1)150• 1150 ,0.6 - - c .§100• .§100• ...... Bo.4 Q.ts 50 50 l0.2

0 0 0.0 no... 1 lH.. 2 lHa. 1 no'a' 2 lHal 1 no... 2

~ Open Arms 15 Closed Arms 10 Open Arm. II I) • L• L• l:-0.1 ~ 10 ~10 ! ld IIJ IIJ o.6 ~ , 0 ~ C L. L. .20.4 5 5 ., ~• ~• E E ls :J :J ~0.2 Z Z a. 0 0 0.0 nola. 1 no... 2 nola. 1 no... 2 lHa. 1 nola. 2

Figure 2. Total time, total entries, proportion oftime, and proportion ofentries into open and closed arms on Trials 1 and 2 ofthe elevated plus-maze. p> .53). There were no test order effects because fear con­ the 1st postshift day (r = .46, p < .01). This correlation ditioning was given last. probably reflects the fact that shifted rats tend to return to the level of an unshifted control group, which repre­ Correlations Between Variables sents performance (intake) due to the absolute reward For purposes of correlational analyses, all variables value of4% sucrose. Given that this is a stable parameter were converted to z scores, and Pearson correlation coef­ ofthe population, all rats would tend to return to the same ficients were computed. The resultant correlation matrix approximate level, and, thus, the rats with a greater shift is shown in Table 2. The data will be presented and dis­ would tend to have a steeper recovery slope. cussed in terms ofwithin-test correlations, then between­ No reliable correlations were found with Day 1 licks test correlations, and, finally, factor analysis. and Day 1 latency to first lick. This failure to find a cor­ relation is informative in that it suggests that whatever Within-Test Correlations neophobic reactions might have been involved with the Several additional variables were used in the correla­ initial exposure to sucrose, this neophobia was not related tion analysis that were not described above. For negative to the degree ofdecrement when the rats were shifted from contrast, Day 1 licks and Day 1 latency were used to model 32% to 4% sucrose. initial responsivity to the sucrose solution. To portray re­ Elevated plus-maze. Variables from the elevated plus­ covery from contrast, the slope ofthe recovery line (lick maze were highly interrelated within a trial. For instance, frequency) from Day 11 to Day 14 was computed. Vari­ time in open arms for Trial 1 and open arm entries for ables for plus-maze, emergence, and fear conditioning Trial 1 were highly correlated (r = .86, p < .01), and the remained the same as described above. same measures were also highly correlated for Trial 2 Negative contrastprocedure. Variables from the neg­ (r = .93, p < .0I). Measures between trials were signifi­ ative contrast procedure were interrelated. Slope of re­ cantly correlated, but not as highly as within-trial mea­ covery was significantly correlated with percent shift on sures. For instance, time in open arms in Trials 1 and 2 (r = .j:>. o tv

'Tj t"' > ::r:: t'I1 El ,to<: C) Gl Table 2 Correlation Matrix for All Variables ~ Trial I Trial2 o Two- Four- Time Open- Proportionof Proportionof Time Open- Proportionof Proportionof Proportion p Percent paw paw in open arm time in open-arm Total in open arm time in open-arm Total of time ~ shift Slope latency latency arms entries open arms entries entries arms entries open arms entries entries freezing > Percentshift El Slope .47* ,Z Two-pawlatency -.04 -.26 Four-paw latency -33* -.22 .55* ~ Trial I o Timein open arms .IS .11 .03 -.26* t"' Open-armentries 31* .25 -.11 -.35* .86* t'I1 Vl Proportion of timeinopenarms .20 .14 .02 -.26* .99* .SS* N Proportionof open-armentries .16 .15 .05 -.04 .79* .S'* .79* o Totalentries .35* .20 -.IS -.52* .50* .71* .49* .25 Nc Trial 2 ~ Timein open arms .02 .08 -.OS -.09 37* .45* .3S· .25* .42* Open-armentries .II .18 -.13 -.15 .42* .50* .42· .32* .44* .93* Proportion of timein openarms .05 .07 -.OS -.10 35* .43* .36· .23 .42* .99* .93* Proportionof open-armentries .06 .14 -.05 -.03 .40* .44* .41· .33* .31* .91* .94* .91* Totalentries .16 -.03 -.14 -.27* 31* .39* .31· .16 .57* .49* .61* .4S* .42* Proportionof time freezing .06 .IS 3S .32 -.03 -.I 7 -.07 .02 -.19 -.IS -.23 -.22 - .21 -.15 *p < .05. CONTRAST AND ANXIETY 403

.37,p < .01) were correlated, as was time in open arms in Elevated plus-maze and emergence. There was a Trial 1 and open-arm entries in Trial 2 (r = 042, p < .01). substantial relationship between four-paw emergence la­ Although there were some reliable between-trial cor­ tency and Trial 1 performance in the elevated plus-maze. relations, percent ofvariance explained by the correlations Generally, long emergence times were related to hesitancy was higher for within-trial variables than for between­ to enter the open arms in the plus-maze (latency and num­ trial variables. This finding is consistent with earlier re­ ber ofopen arm entries on Trial 1, r = - .35,p < .01; la­ ports that Trial 1 and Trial 2 reflect different types offear tency and time spent in open arms on Trial 1, r = - .26, (File et aI., 1993). p < .05; latency and proportion oftime spent in open arms Emergence. Latency to emerge with two paws was on Trial 1, r = - .26, p < .04; latency and total entries on positively correlated with latency to emerge with four Trial 1, r = -.51,p < .01). paws (r = .55, P < .01). However, there was substantial Total number of entries was the only Trial 2 variable variance unexplained by this correlation (r2 = .30), which correlated with four-paw emergence (r = - .27, P < .05). suggests that there is something quite different about these The separation seen between Trial 1 and Trial 2 behav­ two measures ofemergence. ior in this test supports the previous data and File's earlier statement that Trials 1 and 2 measure different psycho­ Between-Test Correlations logical phenomena (File et aI., 1993). The finding that Negative contrast procedure and elevated plus­ number oftotal entries was the only Trial 2 variable to cor­ maze. Percent shift correlated positively with Trial 1 relate with emergence suggests that a factor related to gen­ open-arm entries (r = .31, p < .02) and Trial 1 total en­ eral activity might be something in common between the tries (r = .35,p < .01). The positive correlation between two trials in the plus-maze and the emergence test. degree of shift and frequency of arm entries may be Emergence and conditioned fear. There was a sig­ taken as support for the hypothesis that an early stage of nificant positive correlation found between proportion of contrast involves search for the "missing" preshift re­ time spent freezing and two-paw latency (r = .36, p < ward (Flaherty et aI., 1990) and that rats that show larger .05). The correlation between four-paw latency and time contrast may be the ones less inhibited in regard to leav­ spent freezing came close to reliability (r = .32,p < .08). ing the familiar and exploring the novel. Given the relationship between degree ofcontrast and Factor Analysis Trial 1 behavior, the lack of a correlation between con­ The data were submitted to a factor analysis, using trast and Trial 2 open-arm entries (r = .11, p > AI) as well a principal component solution with an oblique rotation as total Trial 2 entries (r = .16, p > .24) is consistent with of the factor matrix (promax). An oblique rotation was the hypotheses that different psychological processes are selected on the assumption that the factors underlying involved in Trial 1 and Trial 2 behavior in the elevated the various tests of emotionality employed here would plus-maze. have some degree of correlation, as suggested by the The failure ofthe proportion measures ofTrial 1 per­ finding that benzodiazepine tranquilizers are effective in formance to correlate with shift behavior (proportion of time spent in open arms, r = - .20, p > .10; proportion of Table 3 entries into open arms, r = - .16, p > .23) may indicate Final Rotated (Oblique) Factor Pattern that the important factor related to contrast is activity or Factor exploration in this apparatus rather than a specific lack I 4 offear ofthe open arms. 2 3 The failure ofthe slope ofthe recovery function to cor­ Percent shift -.162 -.087 .040 .847* Recovery slope -.084 .180 -.336 .921* relate with any measures from the plus-maze suggests Two-paw latency -.044 -.025 .910* -.221 that it is the initial occurrence ofcontrast, rather than the Four-paw latency .007 -.099 .896* -.148 recovery process, that is related to behavior in the elevated Trial 1 plus-maze. Time in open arms .332 .943* -.140 -.017 Open-arm entries .270 .943* -.378 .127 Negative contrastprocedureand emergence. Greater Proportion oftime .334 .952* -.172 .044 degrees ofcontrast were related to relatively rapid emer­ in open arms gence into the open field (four-paw latency)-a result Proportion ofopen-arm .107 .884* .012 .032 consistent with the relationship between contrast and entries Total entries .342 .557* -.719* .249 Trial 1 elevated plus-maze performance in suggesting that Trial 2 larger degrees ofcontrast are related with a tendency to Time in open arms .969* .226 -.097 -.188 enter novel environments. Open-arm entries .967* .367 -.233 -.088 Negative contrast procedure and conditioned fear. Proportion oftime .958* .163 -.064 -.179 The correlational data indicated no relationship between in open arms Proportion ofopen-arm .937* .332 -.028 -.026 these tests on the 1st postshift day. entries Elevated plus-maze and conditioned fear. No vari­ Total entries .577* .021 -.562* -.022 ables from the plus-maze were correlated with condi­ Proportion oftime freezing -.113 -.140 .478* .298 tioned fear. *Factor loadings greater than ± .45. 404 FLAHERTY, GREENWOOD, MARTIN, AND LESZCZUK

aspects ofall ofthe tests (Davis, 1991;File, 1991; Flaherty, Table 5 1991b). Interpretation of the Factor Pattern Four factors were extracted using the default criterion Factor 1: Plus-Maze Trial 2 (Learned Fear ofthe Open Arms) ofan eigenvalue greater than one. These four factors were Time in open arms Number ofopen entries found to account for 91% ofthe variance. The final rotated Proportion oftime spent in open arms factor pattern is shown in Table 3. Only loadings greater Proportion ofopen entries than ±.45 were considered meaningful (Floyd & Wida­ Total entries man, 1995), and these are highlighted in the table. Factor 2: Plus-Maze Trial 1 (Generalized Anxiety State) Because Day I licks and Day 1 latency were not sig­ Time in open arms nificantly correlated with any measures, these variables Number ofopen entries were dropped from the factor analysis. As seen in Table 4, Proportion oftime spent in open arms Proportion ofopen entries plus-maze variables were loaded on the first two factors: Total entries Trial 2 variables loaded on Factor 1 (accounting for the Factor 3: Emergence, Context-Shock Conditioning (Timidity/Fear) most variance in the set), whereas Trial 1 variables Two-paw latency loaded on Factor 2. Emergence and fear conditioning vari­ Four-paw latency ables loaded on Factor 3, whereas negative contrast loaded Proportion oftime spent freezing alone on Factor 4. Total entries, Trial 1* The final factor intercorrelations are presented in Ta­ Total entries, Trial 2* ble 4. Factor 1 and Factor 2 (the two plus-maze factors) Factor 4: Successive Negative Contrast (Disappointment) Percent shift were more highly correlated than any other pair of fac­ Slope ofrecovery function tors. Factors 3 and 4 shared relatively little common vari­ *Negative loading. ance with the other factors. An interpretation of the factors is shown in Table 5. Factor I loaded plus-maze Trial 2 variables only. Because Factor 3 loaded latency to emerge from a familiar to an rats show an increased avoidance of the open arms on unfamiliar environment and proportion oftime spent freez­ Trial 2 relative to on Trial I, and because chlordiazepox­ ing in a shock-paired context. This constellation suggests a ide is generally ineffective on Trial 2, File (1995) has commonality in the emotional state (or behavioral reper­ considered Trial 2 performance to be based on learned toire) involved in an apparent innate fear ofemerging from fear of the open arms-a form of "phobia" rather than a familiar place into a novel environment/open space and generalized anxiety or fear ofnovel places. learned fear of an environment paired with shock. The Factor 2 consisted entirely ofTrial 1 behaviors in the finding that total entries measures from Trial 1 and Trial 2 elevated plus-maze. The greater amount oftime that rats ofthe plus-maze loaded negatively on this factor suggests spend in the closed arms represents a fear of openness a role for general activity level in the sense that a tendency (Treit et aI., 1993), which could be related to fear of to be active detracts from inhibition ofbehavior in both in­ predators or fear of intraspecific encounters (Pellow nate and conditioned fear tasks (as measured by these two et aI., 1985; Rodgers & Dalvi, 1997). However, the fact tests, but apparently not the elevated plus-maze). Perhaps that the animals do enter the open arms suggests a con­ there is an underlying dimension ofactivity/inhibition that flict between exploratory tendencies and fear of open modifies performance in these tasks-a dimension related spaces. Exposure to this apparatus activates various phys­ to "timidity" or response inhibition (cf. Gray's, 1987, view iological indices of stress, including increased extra­ ofintroversion vs. extroversion or neuroticism). cellular cortical 5-HT (Rex, Marsden, & Fink, 1994), Negative contrast variables loaded exclusively on Fac­ and corticosterone (File et aI., 1994; Pellow & File, 1986). tor 4. This finding suggests that initial responsivity to re­ Also, open-arm entries on this trial may be increased by ward reduction is distinct from the reactions involved in the administration of benzodiazepines (Pellow & File, the other three tests-a result consistent with interpreta­ 1986). File's interpretation ofTrial 1 in the elevated plus­ tions of initial degree of contrast in terms of detection, maze is that the avoidance ofthe open arms represents a evaluation, and search processes rather than emotionality generalized anxiety state (File et aI., 1993). per se (Flaherty, 1996).

Table 4 Postshift Data Correlations Between the Four Factors Because evidence from drug studies and from measure­ Factor ment ofcorticosterone has indicated that the nature ofneg­ ative contrast changes across the postshift period, further Factor 2 3 4 factor analyses were conducted with percent shift calcu­ 1 2 289 lated for each of the last 3 postshift days. Table 6 illus­ 3 -.143 -.203 trates the dynamic aspect ofnegative contrast as the rats 4 -.118 .068 -.173 recover from reward reduction across the postshift period. Note-See Table 3 for the variables that loaded onto each factor. The loading ofpercent shift on Factor 4 declines across CONTRAST AND ANXIETY 405

Table 6 Animal Models ofAnxiety The Changing Factor Structure Across the Postshift Period Emergence, the elevated plus-maze, negative contrast, Day and fear conditioning are all considered animal models Variable 2 3 4 ofemotion; however, procedurally and empirically, there Factor I would seem to be many differences among these models. Trial 2 Home-cage emergence and the first trial on the elevated Time in open arms .97 .97 .97 .97 plus-maze could reflect unconditioned behaviors. Context­ Open-arm entries .97 .96 .96 .97 shock fear is associative, and negative contrast may also Proportion oftime .96 .96 .96 .96 be associative in that the animals learn to expect a given in open arms Proportion ofopen-arm .94 .93 .92 .93 level ofreward as a function ofprior experience. entries Factor analytic data ofplus-maze performance obtained Total entries .58 .59 .59 .58 in this experiment and in other experiments suggest that Factor 2 the nature ofthe anxiety involved in this test changes with Trial I experience in the apparatus (Fernandes & File, 1996; File, Time in open arms .94 .94 .94 .94 1996; File et aI., 1993; Hogg, 1996; Rodgers et aI., 1996). Open-arm entries .94 .94 .94 .94 The first trial may measure generalized anxiety, a condi­ Proportion oftime .95 .95 .95 .95 tion that may be alleviated by benzodiazepines and other in open arms Proportion ofopen-arm .88 .89 .89 .88 anxiolytic agents. The experience gained on the first trial entries may lead to the development ofa specific fear ofthe open Total entries .56 .55 .55 .55 arms, as evidenced by the decline in open-arm entries Factor 3 from Trial 1 to Trial 2 seen in the present study and in Two-paw latency .91 .91 .91 .88 earlier studies. Support for the hypothesis that Trial 2 rep­ Four-paw latency .90 .87 .86 .88 resents a different type ofanxiety is the finding that ben­ Total entries zodiazepine anxiolytics are effective on Trial 1 but are Trial I -.71 -.73 -.72 -.69 Trial 2 -.56 -.53 -.49 -.56 ineffective on Trial 2 (e.g., File, 1995). Conditioned freezing .48 .31 .27 .58 The data suggest still more facets ofemotional shading Percent shift .04 .16 .26 .45 or behavioral repertoire measured by these tests. For in­ Slope -.34 -.53 -.58 -.24 stance, the separate aspects ofanxiety/fear apparently mea­ Factor 4 sured in Trials 1 and 2 ofthe elevated plus-maze and con­ Percent shift .85 .75 .52 -.80 ditioned fear established by context-shock pairings loaded Slope .92 .46 .52 .81 on three distinct factors. A priori, it would seem that there Conditioned freezing .30 .72 .79 .42 should be a strong relationship between context-conditioned Two-paw latency -.22 .31 .27 -.54 fear and performance on Trial 2 in the plus-maze, given Note-Conditioned freezing and percent shift factor loadings below ±.45 are included to illustrate the changing nature ofthe factors across File's interpretation that Trial 2 represents a learned fear. the postshift period. However, Trial 2 in the plus-maze and shock-conditioned fear loaded on two different factors. Also, there were no re­ liable correlations between context-shock-conditioned fear the postshift period, becoming strongly negative on the and elevated plus-maze performance. Perhaps different un­ last postshift day. Ofgreater interest is the fact that con­ conditioned stimuli (shock vs. openness) condition differ­ ditioned fear and contrast load on the same factors on the ent behaviors/emotions. Because of the design of this ex­ 2nd and 3rd postshift days, when contrast may involve an periment, it is not known whether plus-maze performance emotional reaction, but not on the 1st postshift day, when would have been altered if context-shock fear condition­ corticosterone is not elevated and when chlordiazepox­ ing was given prior to the plus-maze experience. ide and ethanol are not effective in reducing contrast. On Successive negative contrast has long been attributed to the last postshift day, the factor structure is different still: an emotional reaction to the reward reduction, but differ­ contrast now loads with fear on a different factor (Fac­ ent labels have been applied to the postulated emotion in­ tor 3), along with emergence latency and plus-maze ac­ volved: anger (Crespi, 1944; Tinklepaugh, 1928), depres­ tivity measures. Behaviorally, contrast has typically dis­ sion (Crespi, 1942), frustration (Amsel, 1992; Spence, sipated by the 4th postshift day. The factor loadings seen 1956), and disappointment (Flaherty, 1996). Interpretation here may suggest that there is still an underlying emo­ of the correlational and the factor analytic data indicate tional reaction to the shift even though the shifted animals some relationships between negative contrast and other have typically recovered by the last postshift day. tests ofemotionality, but the more striking outcome is the degree of independence of the four tests employed here. DISCUSSION The relationship between the plus-maze and the emer­ gence test is complicated. Although the two tests loaded Two aspects of the data will be considered here: the on distinct factors, there were several reliable correlations relevance of the results to animal models of anxiety in between four-paw latency to emerge and Trial 1 behav­ general, and the implications of the results for interpre­ ior in the plus-maze. Part ofthis relationship may be due tations of SNC. to general activity level, since there were negative corre- 406 FLAHERTY, GREENWOOD, MARTIN, AND LESZCZUK

lations between emergence and total entries on Trials 1 BECKER, H. c., JARVIS, M., WAGNER, G., & FLAHERTY, C. F. (1984). and 2. A part may also be specifically fear-related, be­ Medial and lateral amygdala lesions differentially influence contrast cause there was a negative correlation between emergence with sucrose solutions. Physiology & Behavior, 33,707-712. BELZUNG. C., & LE PAPE, G. (1994). Comparison of different behav­ latency and total open entries on Trial 1 and also between ioral test situations used in psychopharmacology for measurement of emergence latency and proportion of open entries on anxiety. Physiology & Behavior, 56, 623-628. Trial 1. This relationship ofemergence to Trial 1 elevated BLANCHARD, R. J., KELLEY, M. J., & BLANCHARD, D. C. (1974). De­ plus-maze behavior, but not Trial 2, further supports the fensive reactions and exploratory behavior in rats. 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